L-selectin Expression Research Articles

Immune Defense in Upper Airways: A Single-Cell Study of Pathogen-Specific Plasmablasts and Their Migratory Potentials in Acute Sinusitis and Tonsillitis.

BackgroundDespite the high frequency of upper respiratory tract (URT) infections and use of the nasal mucosa as route for vaccination, the local immune mechanism and dissemination of effector lymphocytes from the URT have been insufficiently characterized. To devise a single-cell approach for studying the mucosal immune response in the URT, we explored URT-originating B effector lymphocytes in the circulation of patients with one of two common respiratory infections, acute sinusitis or tonsillitis.MethodsPatients with acute sinusitis (n = 13) or tonsillitis (n = 11) were investigated by ELISPOT for circulating pathogen-specific antibody-secreting cells (ASCs) of IgA, IgG and IgM isotypes approximately one week after the onset of symptoms. These cells’ potential to home into tissues was explored by assessing their expression of tissue-specific homing receptors α4β7, L-selectin, and cutaneous lymphocyte antigen (CLA).ResultsPathogen-specific ASCs were detected in the circulation of all patients, with a geometric mean of 115 (95% CI 46–282) /106 PBMC in sinusitis, and 48 (27–88) in tonsillitis. These responses were mainly dominated by IgG. In sinusitis α4β7 integrin was expressed by 24% of the ASCs, L-selectin by 82%, and CLA by 21%. The proportions for tonsillitis were 15%, 80%, and 23%, respectively. Healthy individuals had no ASCs.ConclusionsURT infections–acute sinusitis and tonsillitis–both elicited a response of circulating pathogen-specific plasmablasts. The magnitude of the response was greater in sinusitis than tonsillitis, but the homing receptor profiles were similar. Human nasopharynx-associated lymphoid structures were found to disseminate immune effector cells with a distinct homing profile.

Activated mast cells promote differentiation of B cells into effector cells.

Based on the known accumulation of mast cells (MCs) in B cell-dependent inflammatory diseases, including rheumatoid arthritis, we hypothesized that MCs directly modulate B cells. We show here that degranulated, and to a lesser extent naïve or IgE-sensitized, MCs activate both naïve and B cell receptor-activated B cells. This was shown by increased proliferation, blast formation, and expression of CD19, MHC class II and CD86 in the B cells. Further, MCs stimulated the secretion of IgM and IgG in IgM+ B cells, indicating that MCs can induce class-switch recombination in B cells. We also show that coculture of MCs with B cells promotes surface expression of L-selectin, a homing receptor, on the B cells. The effects of MCs on B cells were partly dependent on cell-cell contact and both follicular and marginal zone B cells could be activated by MCs. Our findings suggest that degranulated MCs support optimal activation of B cells, a finding that is in line with in vivo studies showing that MCs frequently degranulate in the context of B-cell driven pathologies such as arthritis. Together, our findings show that MCs have the capacity to differentiate B cells to effector cells.

Abstract WP261: Sex Differences in Circulating Leukocyte Subtype and Activation Following Ischemic Stroke

Background and Purpose: Females exhibit a more robust immune response in many disease models, yet sex differences in the inflammatory response to ischemia remain largely unexplored. We conducted flow cytometry and RNA sequencing of blood from ischemic stroke patients, with follow-up studies in mice. Hypothesis: We assessed the hypothesis that there are sex differences in the acute immune response to ischemic stroke. Methods: Patient samples were drawn at 24 hours post-stroke for flow cytometry (n=6) and RNA sequencing Analysis (n=40). For murine studies, male and ovariectomized (OVX) female animals (n=14) were subjected to 90-minute middle cerebral artery occlusion or sham surgery and sacrificed at 24 hours. Murine blood was stained for leukocyte markers and CD62L (L-selectin). Results were analyzed by student T-test and two-way ANOVA. Results: RNA sequencing revealed that 24 hours after ischemic stroke, female patients had 79 significantly upregulated genes, compared to male patients with 6 significantly upregulated genes. Human flow cytometry revealed that male stroke patients had a significantly higher percentage of monocytes (p=.026), while females had a greater percentage of CD8+ T-cells (p=.023). Murine flow cytometry showed a post-stroke increase in peripheral myeloid cells at 24 hours in male mice only (p=.0046), whereas female mice had a higher CD8/CD4 T cell ratio (p=.0027). Neutrophils from male sham animals displayed greater L-selectin positivity, with stroke-induced shedding of L-selectin seen only in males (sex/stroke p=.0266). Male monocytes and lymphocytes also displayed higher L-selectin positivity (p=.0079, p=.0004). Conclusion: These results suggest that the immune response to ischemic stroke is different in male and female patients, a phenomenon that can be recapitulated in a mouse model of experimental stroke. Female immune cells exhibit a higher level of baseline activation (reduced L-selectin expression) and post-stroke activity, which may enable a quicker and more robust response to immune challenges. Understanding sex differences in the acute immune response is crucial to developing future immunomodulatory drugs for the safe and effective treatment of ischemic stroke in both sexes.

L-selectin Is Essential for Delivery of Activated CD8(+) T Cells to Virus-Infected Organs for Protective Immunity.

SummaryCytotoxic CD8+ T lymphocytes play a critical role in the host response to infection by viruses. The ability to secrete cytotoxic chemicals and cytokines is considered pivotal for eliminating virus. Of equal importance is how effector CD8+ T cells home to virus-infected tissues. L-selectin has not been considered important for effector T cell homing, because levels are low on activated T cells. We report here that, although L-selectin expression is downregulated following T cell priming in lymph nodes, L-selectin is re-expressed on activated CD8+ T cells entering the bloodstream, and recruitment of activated CD8+ T cells from the bloodstream into virus-infected tissues is L-selectin dependent. Furthermore, L-selectin on effector CD8+ T cells confers protective immunity to two evolutionally distinct viruses, vaccinia and influenza, which infect mucosal and visceral organs, respectively. These results connect homing and a function of virus-specific CD8+ T cells to a single molecule, L-selectin.

Significance of TNF-α and the Adhesion Molecules: L-Selectin and VCAM-1 in Papillary Thyroid Carcinoma.

Circulating levels of TNF-α and the adhesion molecules L-Selectin and VCAM-1 as well as their expression in the primary tumors of patients with benign thyroid diseases and papillary thyroid carcinoma (PTC) have been determined in this study. The serum levels of TNF-α, L-Selectin, and VCAM-1 were significantly higher in patients with both benign thyroid diseases and PTC as compared to the healthy individuals. However, the levels of only TNF-α and L-Selectin, and not VCAM-1, were significantly higher in patients with PTC in comparison to those observed in patients with benign thyroid diseases. Further the expression of TNF-α and L-Selectin was also significantly higher in the primary tumors of PTC patients, relative to the benign thyroid diseases. The expression of L-Selectin and VCAM-1 significantly correlated with aggressive tumor behavior. In PTC patients, the circulating TNF-α levels significantly positively correlated with the levels of L-Selectin, while TNF-α immunoreactivity was significantly associated with VCAM-1 expression. Serum TNF-α was found to be a significant prognosticator for OS in PTC patients. Overall the results signify that the interaction between TNF-α and the adhesion molecules may have a role in thyroid carcinogenesis and understanding this complexity may offer potential therapeutic targets for better management of thyroid cancer.

Freeze and Thaw of CD4+CD25+Foxp3+ Regulatory T Cells Results in Loss of CD62L Expression and a Reduced Capacity to Protect against Graft-versus-Host Disease.

The adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs) in murine models of allogeneic hematopoietic cell transplantation (HCT) has been shown to protect recipient mice from lethal acute graft-versus-host disease (GVHD) and this approach is being actively investigated in human clinical trials. Here, we examined the effects of cryopreservation on Tregs. We found that freeze and thaw of murine and human Tregs is associated with reduced expression of L-selectin (CD62L), which was previously established to be an important factor that contributes to the in vivo protective effects of Tregs. Frozen and thawed murine Tregs showed a reduced capacity to bind to the CD62L binding partner MADCAM1 in vitro as well as an impaired homing to secondary lymphoid organs in vivo. Upon adoptive transfer frozen and thawed Tregs failed to protect against lethal GVHD compared with fresh Tregs in a murine model of allogeneic HCT across major histocompatibility barriers. In summary, the direct administration of adoptively transferred frozen and thawed Tregs adversely affects their immunosuppressive potential which is an important factor to consider in the clinical implementation of Treg immunotherapies.

Effects of prepartum stocking density on innate and adaptive leukocyte responses and serum and hair cortisol concentrations

Objectives were to evaluate the effects of prepartum stocking density on innate and adaptive leukocyte responses, serum cortisol and haptoglobin concentrations and hair cortisol concentration of Jersey cows. The cows (254±3d of gestation) were balanced for parity (nulliparous vs. parous) and previous lactation projected 305-d mature equivalent milk yield and assigned to one of two treatments: 80SD=80% stocking density (38 animals/48 headlocks) and 100SD=100% stocking density (48 animals/48 headlocks). Pens (n=4) were identical in size and design and each pen received each treatment a total of 2 times (4 replicates; 80SD: n=338; 100SD: n=418). A sub-group of cows (n=48/treatment per parity) was randomly selected on week 1 of each replicate from which blood was sampled weekly from d −14 to 14 (d 0=calving) to determine polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of CD18 and L-selectin, and hemogram. The same sub-group of cows was treated with chicken egg ovalbumin on d −21, −7, and 7 and had blood sampled weekly from d −21 to 21 for determination of serum IgG anti-ovalbumin concentration. Blood was sampled weekly from d −21 to 21 to determine glucose, cortisol, and haptoglobin concentrations in serum. Hair samples collected at enrollment and within 24h of calving were analyzed for cortisol concentration. The percentage of leukocytes classified as granulocyte and the granulocyte to the lymphocyte ratio were not affected by treatment. Treatment did not affect the percentage of PMNL positive for phagocytosis and oxidative burst or the intensity of phagocytosis and oxidative burst. Similarly, treatment did not affect the percentage of PMNL expressing CD18 and L-selectin or the intensity of expression of CD18 and L-selectin. Concentration of IgG anti-ovalbumin was not affected by treatment. Serum concentrations of haptoglobin and cortisol were not affected by treatment. Similarly, hair cortisol concentration at calving was not affected by treatment. According to the current experiment, a target stocking density of 80% did not improve leukocyte responses compared with 100% target stocking density.

Abstract IA23: T cell trafficking in lymphoid and non-lymphoid tissues

Abstract Increased representation of CD8 T-lymphocytes in tumors, spontaneously, or after vaccination or adoptive therapy, is essential for immune mediated control of human cancers. T-cell infiltrates are a positive prognostic indicator of patient survival, and it has become clear that the patients who respond clinically to new generation immunotherapies are those in which an immunological infiltrate is already evident prior to treatment. Thus, limited representation of intratumoral T-cells is a major barrier to tumor control, distinct from the barrier created by immunosuppression. Understanding and overcoming this limitation will enable us to extend the effectiveness of many immunotherapies to a broader cross-section of cancer patients. T cell infiltration into lymphoid and non-lymphoid tissues is controlled by homing receptors (HR) that engage corresponding ligands expressed on tissue-associated vasculature. Naïve T-cells enter LN based on expression of L-selectin and CCR7. These HR bind to peripheral node addressin (PNAd) and the chemokines CCL19/CCL21, respectively, which are expressed on specialized lymph node blood vessels called high endothelial venules (HEV). During differentiation, effector T-cells acquire the ability enter peripheral tissues, including tumors, by upregulating other selectins, integrins and chemokine receptors that bind to ligands expressed on inflamed vasculature. However, we lack a full understanding of which HR expressed by CD8 T-cell effectors control their migration into tumors, how this is influenced by expression of the corresponding ligands on tumor vasculature, and whether ligand expression changes as a result of vessel normalization. We have explored the hypothesis that CD8 T-cell effectors activated in different secondary lymphoid organs express distinct HR, which in turn control infiltration into tumors in different anatomical locations. We identified 3 major populations of primary CD8 T-cell effectors based on site of activation and HR expression pattern, and have established which HR enable infiltration of one of these populations into tumors. While it has been assumed that all tumor-infiltrating lymphocytes are effectors that differentiate in tumor-draining LN and subsequently enter the tumor, we have shown that naïve CD8 T-cells also can directly enter tumors. These naïve cells differentiate into functional effectors in the tumor, and can promote its control. Naïve CD8 T-cell infiltration depends on L-selectin and CCR7, as in lymph nodes, and on the development of blood vessels in the tumor that express PNAd and CCL21 and resemble lymph node HEV. Similar lymph node-like vasculature has been reported in human tumors along with ectopic organized lymphoid tissue (tertiary lymphoid organs or TLO), and correlated with longer metastasis-free, disease-free, and overall survival. Development of this vasculature in murine tumors was controlled by a novel mechanism involving effector CD8 T-cells and NK cells that secreted Lymphotoxin-α3 and IFNγ;. This suggests that early infiltration of effectors induces LN-like vasculature, which in turn supports a self-sustaining recruitment of naïve T-cells that differentiate within the tumor. In intraperitoneal but not subcutaneous tumors, this lymph node-like vasculature was also associated with organized aggregates of B-lymphocytes and gp38+ fibroblasts that resembled TLO. The differential expression of these elements in SC and IP tumors reflects differences in immunological tumor microenvironments that remain to be explored further. Nonetheless, these results establish LN-like vasculature as both a consequence of and key contributor to anti-tumor immunity based on its role in promoting T-cell infiltration. Citation Format: Victor H. Engelhard, David Peske, Amber Woods, Nithya Srinivasan, Colin Brinkman, Andrew Ferguson. T cell trafficking in lymphoid and non-lymphoid tissues. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr IA23.

L-selectin controls trafficking of chronic lymphocytic leukemia cells in lymph node high endothelial venules in vivo

AbstractB-cell chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Lymph nodes (LNs) are sites of malignant proliferation and LN enlargement is associated with poor prognosis in the clinics. The LN microenvironment is believed to favor disease progression by promoting CLL cell growth and drug resistance. A better understanding of the mechanisms regulating trafficking of CLL cells to LNs is thus urgently needed. Here, we studied the first step of CLL cell migration to LNs, their interaction with high endothelial venules (HEVs), specialized blood vessels for lymphocyte extravasation in lymphoid organs. We observed that the density of HEV blood vessels was increased in CLL LNs and that CD20+ CLL cells accumulated within HEV pockets, suggesting intense trafficking. We used intravital imaging to visualize the behavior of human CLL cells within the mouse LN microcirculation, and discovered that CLL cells bind to HEVs in vivo via a multistep adhesion cascade, which involves rolling, sticking, and crawling of the leukemic cells on the endothelium. Functional analyses revealed that the lymphocyte homing receptor L-selectin (CD62L) is the key factor controlling the binding of CLL cells to HEV walls in vivo. Interestingly, L-selectin expression was decreased on CLL cells from patients treated with idelalisib, a phosphoinositide-3-kinase δ inhibitor recently approved for CLL therapy. Interference with L-selectin–mediated trafficking in HEVs could represent a novel strategy to block dissemination of CLL cells to LNs and increase the efficacy of conventional therapy.

Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.

HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules. The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate-coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates. HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen. Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.

The effect of kisspeptin on the functional characteristics of isolated NK cells

The effect of kisspeptin at concentrations typical of pregnancy on the functional activity of isolated cytokine-primed NK cells has been investigated. The hormone has been shown to promote an increase in the proportion of CD56(bright) NK cells, as well as an increase in the L-selectin expression on the cell surface. Assessment of cytokine levels has shown that kisspeptin suppresses the production of IL-4, IL-10, and IFN-γ while stimulating the production of TGF-β by isolated NK cells. The overall effect of the hormone investigated consisted in the formation of a phenotype and a cytokine spectrum characteristic of the regulatory NK3 subpopulation of NK cells in pregnancy.

Characterizing the hematopoietic stem/progenitor cells in the placenta and umbilical cord blood from normal and preeclamptic subjects

Homocysteine lowering with folic acid has been shown to be ineffective in reducing vascular events in patients with established cardiovascular disease. However, it may be beneficial in the early stages of atherosclerosis particularly for the population at risk. We have investigated the effect of folic acid administration on homocysteine levels, endothelial function, and leukocyte adhesion molecules in atorvastatin-treated patients. Thirty-nine patients were randomized to folic acid 5 mg daily or no additional treatment. Plasma total homocysteine levels were determined by high-performance liquid chromatography. Biochemical and hematologic tests were performed using routine automated analyzer methods. We examined the expression of l-selectin (CD62L); α-subunits of integrins LFA-1 (CD11a), Mac-1 (CD11b), and VLA-4 (CD49d); and β-subunit of β-2 integrins (CD18) by an immunofluorescence method using single-step staining of whole blood with monoclonal antibodies. The fluorescence was quantified by flow cytometry. Reactivity of the forearm skin microvascular bed to thermal and ischemic stimuli was measured using laser Doppler flowmetry. Postocclusive and thermal reactive hyperemia tests were performed. Statistical analysis used 2-sample t test and paired t test. Homocysteine levels decreased significantly in the folic acid–treated group (11.78 ± 2.7 vs 9.75 ± 1.89 μmol/L, P < .0001) but did not change in the control group (11.17 ± 2.09 vs 11.88 ± 2.88 μmol/L). There was no effect of folic acid treatment on leukocyte expression of cell adhesion molecules or microvascular reactivity. The lack of effect of folic acid on microvascular reactivity and leukocyte adhesion molecules supports the evidence against the beneficial effects of folic acid administration in the early stages of atherogenesis.

Perivascular mast cells regulate vein graft neointimal formation and remodeling.

Objective. Emerging evidence suggests an important role for mast cells in vein graft failure. This study addressed the hypothesis that perivascular mast cells regulate in situ vascular inflammatory and proliferative responses and subsequent vein graft neointimal lesion formation, using an optimized local mast cell reconstitution method.Methods and Results. Neointimal hyperplasia was induced by insertion of a vein graft into the right carotid artery in wild type and mast cell deficient KitW−sh/W−sh mice. In some experiments, mast cells were reconstituted systemically (tail vein injection of bone marrow-derived mast cells) or locally (directly into the right neck area) prior to vein grafting. Vein graft neointimal lesion formation was significantly (P < 0.05) reduced in KitW−sh/W−sh mice. Mast cell deficiency reduced the number of proliferating cells, and inhibited L-selectin, CCL2, M-CSF and MIP-3α expression in the vein grafts. Local but not systemic mast cell reconstitution restored a perivascular mast cell population that subsequently promoted neointimal formation in mast cell deficient mice.Conclusion. Our data demonstrate that perivascular mast cells play a key role in promoting neointima formation by inducing local acute inflammatory and proliferative responses. These results suggest that ex vivo intraoperative targeting of mast cells may have therapeutic potential for the prevention of pathological vein graft remodeling.

Natalizumab treatment reduces L-selectin (CD62L) in CD4+ T cells.

BackgroundThe purpose of this research was to validate the low expression of L-selectin (CD62L) in natalizumab (NTZ)-treated patients. CD62L is involved in rolling and transmigration of leukocyte cells. A correlation between CD62LCD4+ T cells low expression and progressive multifocal leukoencephalopathy (PML) development has been suggested in multiple sclerosis (MS) patients treated with NTZ.MethodsWe performed a flow cytometric analysis on peripheral blood mononuclear cells (PBMC); we collected from 23 healthy donors and 225 MS patients: untreated (n = 19) or treated with NTZ (n = 113), interferon-beta (n = 26), glatiramer acetate (n = 26), fingolimod (n = 23) and rituximab (n = 18). We have also analysed two PML/IRIS (immune reconstitution inflammatory syndrome) patients and four longitudinal samples of a NTZ-treated patients before and during the development of a clinical asymptomatic magnetic resonance imaging (MRI) lesion confirmed as PML by cerebrospinal fluid (CSF) examination. Thirty-five NTZ-treated patients were studied longitudinally with three samples taken 4 months apart.ResultsThe NTZ-treated patients showed a lower percentage of CD62L (33.68 %, n = 113) than first-line treated patients (44.24 %, n = 52, p = 0.0004). NTZ effect was already clear during the first year of treatment (34.68 %; p = 0.0184); it persisted in the following years and disappeared after drug withdrawal (44.08 %). Three percent of longitudinally analysed patients showed a percentage of CD62LCD4+ T cells under a hypothetical threshold and one patient with asymptomatic PML belongs to a group which expressed low percentage of CD62LCD4+ T cells.ConclusionsOur research confirms that NTZ has a specific effect on CD62LCD4+ T cells consisting in decreasing of the number of positive cells. The low level of CD62L found in a clinically asymptomatic PML patient strengthens its potential usefulness as a biomarker of high PML risk in NTZ-treated patients. A larger study is required to better confirm the data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0365-x) contains supplementary material, which is available to authorized users.

Astragalus polysaccharide promotes the release of mature granulocytes through the L-selectin signaling pathway.

BackgroundThis study aims to investigate the leukogenic effect of astragalus polysaccharide (APS), to compare its effect of increasing the numbers of mature granulocytes with that of granulocyte colony-stimulating factor (G-CSF), and to investigate the mechanism.MethodsRats were arbitrarily grouped into four groups (control, cyclophosphamide (CTX), CTX + APS, and CTX + G-CSF groups), and each group was then arbitrarily divided into five subgroups according to the time period since CTX infusion (0, 4, 7, 10, and 14 days). The expression of leukocyte selectin (L-selectin), its ligand, and shedding-related protease on granulocytes was analyzed. Leukocyte counts were obtained. Chemotactic capacity of polymorphonuclear leukocytes (PMNLs) was assessed.ResultsBoth APS and G-CSF restored the expression of L-selectin, P-selectin glycoprotein ligand-1 (PSGL-1), CD11b/CD18, and ADAM17 to normal levels (P > 0.05 vs. control group on each time point), with APS eliciting a greater effect than G-CSF (P = 0.005 on day 7, P < 0.001 on day 10 and 14 for L-selectin; P = 0.038 on day 7, P = 0.001 on day 10, P < 0.001 on day 14 for PSGL-1; P < 0.001 on day 7, 10 and 14 for ADAM17; P < 0.001 on day 7, 10, and 14 for CD11b/CD18). The percentages of the bands and segmented bone marrow (BM) cells in myeloid neutrophils were higher in the CTX + APS group than in the CTX group on day 7 (P = 0.030) and reached normal levels on day 10 (P = 0.547) and 14 (P = 0.431) vs. control group. The ability of APS to increase numbers of PMNLs in peripheral blood after chemotherapy was significantly superior to that of G-CSF 7 days after chemotherapy (P = 0.029 on day 10, P = 0.006 on day 14). Moreover, APS more significantly improved the chemotactic ability of PMNLs among mature BM granulocytes and peripheral blood neutrophils after chemotherapy than did G-CSF (P < 0.001 on day 7, P = 0.001 on day 10 and P = 0.005 on day 14).ConclusionsAPS promoted the differentiation and chemotactic ability of BM granulocytes via the L-selectin signaling pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-015-0043-z) contains supplementary material, which is available to authorized users.

Relationship between expression of L-selectin, integrin αL and integrin β2 and multiple-organ metastasis of breast cancer in TA2 mice

Objective:To investigate the effect of the L-selectin,integrin αL and integrin β2 expressions on the multiple -organ metastasis of breast cancer of TA2 mice.Methods:The pathologic character of spontaneous breast cancer with multiple-organ metastasis (high metastasis,Group A) and without distant metastasis (low metastasis,Group B) were observed by macroscopy and HE staining sections.The level of the L-selectin,integrin αL and integrin β2 expressions in Group A,Group B and the metastatic tumors of Group A were detected using the immunohistochemical method.The level of the mRNA of L-selectin,integrin aL and integrin β2 relative expressions in the prmary tumors of Group A and Group B were detected by Real-time PCR method.Results:Hemorrhage and necrosis were visibly seen in all the primary tumors of the Group A which were poorly differentiated or undifferentiated.Pulmonary,hepatic and splenic metastases,and angiogenetic mimicry were observed in these tumors.The average positive cell percentage of integrin αL and L-selectin in primary tumors of Group A and Group B were 24.81% vs.10.47% and 39.81% vs.16.75%,respectively.The expressions of the proteins were significantly higher in Group A than in Group B (Z=-2.31,-2.14,respectively;P0.05),while there was no significant difference in the expression of integrin (32 between the two groups.In Group A,the average levels of L-selectin expression were 39.81%,39.07%,62.09% and 55.51% in the primary tumors,and the pulmonary,hepatic and splenic metastatic tumors,respectively,among which the average expression of L-selectin was significantly higher in liver metastatic tumor than in primary tumor (Z=-2.24,P=0.025),while there was no markedly difference in the expression of integrin αL and integrin β2 between the primary and metastastic tumor.The levels of L-selectin and integrin αL mRNA relative expression were significantly higher in the primary tumros of Group A than in those of Group B (Z=-3.32,-3.47;P 0.01).Conclusion:L-selectin, integrin αL and integrin β2 could be expressed by spontaneous breast cancer cells of TA2 mice and the expression of L-selectin might be related to liver metastasis.

Effects of recombinant bovine somatotropin during the periparturient period on innate and adaptive immune responses, systemic inflammation, and metabolism of dairy cows

The aim of this experiment was to determine effects of treating peripartum dairy cows with body condition score ≥3.75 with recombinant bovine somatotropin (rbST) on immune, inflammatory, and metabolic responses. Holstein cows (253±1d of gestation) were assigned randomly to 1 of 3 treatments: untreated control (n=53), rbST87.5 (n=56; 87.5mg of rbST), and rbST125 (n=57; 125mg of rbST). Cows in the rbST87.5 and rbST125 treatments received rbST weekly from −21 to 28d relative to calving. Growth hormone, insulin-like growth factor 1, haptoglobin, tumor necrosis factor α, nonesterified fatty acids, β-hydroxybutyrate, glucose, and cortisol concentrations were determined weekly from −21 to 21d relative to calving. Blood sampled weekly from −14 to 21d relative to calving was used for hemogram and polymorphonuclear leukocyte (PMNL) expression of adhesion molecules, phagocytosis, and oxidative burst. Cows were vaccinated with ovalbumin at −21, −7, and 7d relative to calving, and blood was collected weekly from −21 to 21d relative to calving to determine IgG anti-ovalbumin concentrations. A subsample of cows had liver biopsied −21, −7, and 7d relative to calving to determine total lipids, triglycerides, and glycogen content. Growth hormone concentrations prepartum (control=11.0±1.2, rbST87.5=14.1±1.2, rbST125=15.1±1.3ng/mL) and postpartum (control=14.4±1.1, rbST87.5=17.8±1.2, rbST125=21.8±1.1ng/mL) were highest for rbST125 cows. Cows treated with rbST had higher insulin-like growth factor 1 concentrations than control cows (control=110.5±4.5, rbST87.5=126.2±4.5, rbST125=127.2±4.5ng/mL) only prepartum. Intensity of L-selectin expression was higher for rbST125 than for control and rbST87.5 cows [control=3,590±270, rbST87.5=3,279±271, rbST125=4,371±279 geometric mean fluorescence intensity (GMFI)] in the prepartum period. The PMNL intensities of phagocytosis (control=3,131±130, rbST87.5=3,391±133, rbST125=3,673±137 GMFI) and oxidative burst (control=9,588±746, rbST87.5=11,238±761, rbST125=12,724±781 GMFI) were higher for rbST125 cows than for control cows during the prepartum period. Concentrations of serum IgG anti-ovalbumin tended to be higher for rbST125 cows than for control cows (control=0.75±0.11, rbST87.5=0.94±0.10, rbST125=1.11±0.11 optical density) in the prepartum period. Haptoglobin concentration was significantly reduced 7d postpartum for rbST125 treatment compared with control and rbST87.5 treatments (control=2.74±0.28, rbST87.5=2.81±0.28, rbST125=1.87±0.28 optical density). Although treatment tended to affect postpartum β-hydroxybutyrate (control=747.5±40.2, rbST87.5=753.2±40.1, rbST125=648.8±39.7 µmol/L), it did not affect liver contents of total lipids, triglycerides, or glycogen. Incidence of metritis among rbST125 cows was reduced compared with that in control cows (control=23.1, rbST87.5=18.0, rbST125=7.8%). Treatment of dairy cows with 125mg of rbST improved innate immune responses and IgG concentration, with limited effects on metabolism.

Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia.

Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS) under spinal anaesthesia (mepivacaine, sufentanil), epidural anaesthesia (ropivacaine or bupivacaine, sufentanil) or general anaesthesia (ketamine, thiopental, succinylcholine). Chemokinesis was higher (p=0.008) in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02). After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.

Differences in Homing Potentials of Streptococcus pneumoniae-Specific Plasmablasts in Pneumococcal Pneumonia and After Pneumococcal Polysaccharide and Pneumococcal Conjugate Vaccinations.

Background. Mucosal immune mechanisms in the upper and lower respiratory tracts may serve a critical role in preventing pneumonia due to Streptococcus pneumoniae. Streptococcus pneumoniae–specific plasmablasts presumably originating in the lower respiratory tract have recently been found in the circulation in patients with pneumonia. The localization of an immune response can be evaluated by exploring homing receptors on such plasmablasts, yet no data have thus far described homing receptors in pneumonia.Methods. The expression of α4β7, L-selectin, and cutaneous lymphocyte antigen (CLA) on S. pneumoniae–specific plasmablasts was examined in patients with pneumonia (n = 16) and healthy volunteers given pneumococcal polysaccharide vaccine (PPV; n = 14) or pneumococcal conjugate vaccine (PCV; n = 11).Results. In patients with pneumonia, the proportion of S. pneumoniae–specific plasmablasts expressing L-selectin was high, the proportion expressing α4β7 was moderate, and the proportion expressing CLA was low. The homing receptor α4β7 was expressed more frequently in the pneumonia group than in the PPV (P = .000) and PCV (P = .029) groups, L-selectin was expressed more frequently in the PPV group than in the PCV group (P = .014); and CLA was expressed more frequently in the pneumonia group than in the PPV group (P = .001).Conclusions. The homing receptor profile in patients with pneumonia was unique yet it was closer to that in PCV recipients than in PPV recipients. These data suggest greater mucosal localization for immune response in natural infection, which is clinically interesting, especially considering the shortcomings of vaccines in protecting against noninvasive pneumonia.

N-Formyl peptides drive mitochondrial damage associated molecular pattern induced neutrophil activation through ERK1/2 and P38 MAP kinase signalling pathways

Traumatic injury results in a systemic inflammatory response syndrome (SIRS), a phenomenon characterised by the release of pro-inflammatory cytokines into the circulation and immune cell activation. Released from necrotic cells as a result of tissue damage, damage associated molecular patterns (DAMPs) are thought to initiate the SIRS response by activating circulating immune cells through surface expressed pathogen recognition receptors. Neutrophils, the most abundant leucocyte in human circulation, are heavily implicated in the initial immune response to traumatic injury and have been shown to elicit a robust functional response to DAMP stimulation.Here, we confirm that mitochondrial DAMPs (mtDAMPs) are potent activators of human neutrophils and show for the first time that signalling through the mitogen-activated-protein-kinases p38 and extracellular-signal-related-kinase 1/2 (ERK1/2) is essential for this response. At 40 and/or 100μg/ml, mtDAMPs activated human neutrophils, indicated by a significant reduction in the surface expression of l-selectin, and triggered a number of functional responses from both resting and tumour necrosis factor-α primed neutrophils, which included reactive oxygen species (ROS) generation, degranulation, secretion of interleukin-8 and activation of p38 and ERK1/2 MAPKs. Pre-treatment of neutrophils with Cyclosporin H, a selective inhibitor of formyl peptide receptor-1 (FPR-1), significantly inhibited mtDAMP-induced l-selectin shedding as well as p38 and ERK1/2 activation, suggesting that N-formyl peptides are the main constituents driving mtDAMP-induced neutrophil activation. Indeed, no evidence of l-selectin shedding or p38 and ERK1/2 activation was observed in neutrophils challenged with mitochondrial DNA alone. Interestingly, pharmacological inhibition of p38 or ERK1/2 either alone or in combination significantly inhibited l-selectin shedding and IL-8 secretion by mtDAMP-challenged neutrophils, revealing for the first time that MAPK activation is required for mtDAMP-induced neutrophil activation and function.Our findings demonstrate that signalling through FPR-1 and activation of p38 and ERK1/2 MAPKs are key events in mtDAMP-induced neutrophil activation. Gaining an understanding of the signalling pathways involved in mtDAMP-induced neutrophil activation may assist in the development of future therapeutic strategies aimed at targeting the SIRS response to improve the outcome of the hospitalised trauma patient. Reducing the severity of the inflammatory response may realise substantial benefits for the severely injured trauma patient.